Insulin has been found to be present in a wide variety of rat and human tissues in concentrations higher than plasma. The insulin present in extracts of rat liver was shown to be genuine insulin by its elution pattern on Sephadex chromatography, by serial dilution in the insulin radioimmunoassay, by its activity in the glucose oxidation bioassay and by neutralization of bioactivity with anti-insulin antibodies. Liver insulin (like brain insulin) did not change with fasting or with streptozotocin treatment in the rat, or in the ob/ob mouse, conditions associated with markedly altered insulin secretion and receptor content. Genuine insulin was also detected and characterized in IM-9 cultured human lymphocytes and in human fibroblasts in concentrations similar to tissue levels and as much as 50 to 100 times the insulin concentration in the media. Through the use of a monoclonal anti-beef insulin antibody, the insulin in the cells resembles human insulin more than it does beef insulin, the insulin in the media in which they were grown. The results suggest that a good part if not all of the insulin bound to the extrapancreatic tissues may be synthesized in the tissues themselves.